Glycosaminoglycans (GAGs) are important components of the extracellular matrix and play important regulatory roles in cell and tissue physiology and pathophysiology. GAGs are long unbranched polysaccharides consisting of a repeating disaccharide unit. The repeating unit consists of a hexose (six-carbon sugar) or a hexuronic acid, linked to a hexosamine (six-carbon sugar containing nitrogen). GAGs form an important component of connective tissues. GAG chains may be covalently linked to a protein to form proteoglycans. Proteoglycans and collagen are the chief structural elements of all connective tissues. Their synthesis is essential for proper maintenance and repair of connective tissues. In vitro, the introduction of glucosamine, a key precursor for GAGs, has been demonstrated to increase the synthesis of collagen and GAGs in fibroblasts. In vivo, topical application of glucosamine has enhanced wound healing. Glucosamine has also exhibited reproducible improvement in symptoms and cartilage integrity in humans with osteoarthritis (L. Bucci, Nutritional Supplement Advisor, July 1992).
The major proteoglycans found in connective tissue such as cartilage are chondroitin sulfate, dermatan sulfate, keratan sulfate and hyaluronic acid (also known as hyaluronan or HA). Heparin sulfate is also a proteoglycan, although it is not a component of articular cartilage. Newer names for proteoglycans sometime reference function of the core protein within the molecule found in chondroitin sulfate and keratin sulfate, e.g., aggrecan, a large proteoglycan that aggregates with hyaluronan, or reference location (e.g., decorin (dermatan sulfate), which decorates type I collagen fibrils), or reference primary structure, biglycan which has two glycoaminoglycan chains. Chondrocytes are active cells within the cartilage matrix, which manufacture new collagen and proteoglycan molecules while excreting enzymes, which aid in removal of damaged cartilage and proteoglycans. In other tissue with a high content of extracellular matrix such as tendons, ligaments, subcutaneous connective tissue or bone, tissue-specific cells provide the respective function of synthesizing the appropriate composition of extracellular matrix.
Hyaluronan is an integral part of both synovial fluid and articular cartilage, as exemplary tissues. Within the articular cartilage, hyaluronan provides viscoelastic properties allowing ease of motion between opposing surfaces and increasing compressive resistance. Within the synovium, hyaluronan, as a component of synovial fluid, provides an effective barrier regulating the introduction of plasma components. Under normal conditions, the body will synthesize sufficient amounts of base components to maintain and grow healthy articular cartilage, while limiting the production and release of destructive proteinases, inflammatory mediators and catabolic enzymes.
Hyaluronan or hyaluronic acid is a natural, highly charged, polyanionic molecule composed of alternating units of D-glucuronic and 2-acetamido-2-deoxy-D-glucose. These unbranched, coiled, elongated polysaccharide chains maintain a large negative electrostatic charge that attracts water molecules and allow the deformation of the molecular coil as ice crystallisation occurs during freezing and thawing. It is believed that hyaluronic acid coats and protects cells and tissues by attaching to the CD44 receptor sites on cells. Hyaluronic acid and other complex GAG formulations are frequently administered by intra-articular injection to treat joint disease, including osteoarthritis wherein they improve clinical symptoms and slow disease progression. Another example is the rebuilding of subcutaneous structures with the injection of hyaluronic acid or hyaluronic acid and other complex GAG formulations.
Chondroitin sulfate is broken down into sulfate disaccharides and N-acetyl galactosamine. Chondroitin sulfate, as CS4 and CS6 sulfated forms, within the body, is thought to be an essential glycosaminoglycan that binds water to the articular cartilage matrix and is necessary for the formation of proteoglycans. In particular, chondroitin sulfate is a long hydrophilic chain of repeating sugars. This glycosaminoglycan binds to proteoglycan molecules aiding in water and nutrient transportation within the articular cartilage. Chondroitin in its sulfate form includes galactosamine, a primary substrate of hyaluronan and a disaccharide pathway for proteoglycan synthesis secondary to the hexosamine pathways utilised for glycosaminoglycan production. Chondroitin sulfate chains comprise the space formation of the cartilage matrix and integral parts of the proteoglycan molecule. Chondroitin stimulates the production of proteoglycans, glycosaminoglycans, and collagen, which are the building blocks of healthy cartilage. Chondroitin sulfate also inhibits the secretion of degenerative enzymes by the chondrocytes within articular cartilage. Chondroitin sulfates are non-toxic and work synergistically with glucosamine to hydrate and repair articular cartilage.
Glucosamine is an amino sugar and a precursor for glycosaminoglycans (GAGs). Glucosamine, as glucosamine 5-phosphate, is naturally occurring within the body and is a component in the biosynthesis of glycosaminoglycans, proteoglycans, hyaluronan, and collagen. Glucosamine is available in exogenous forms, glucosamine sulfate sodium, glucosamine hydrochloride and N-acetyl D-glucosamine. N-acetyl D-glucosamine is also a derivative of glucose obtained by chemical hydrolysis of chitin. This polysaccharide is readily soluble in water and extremely bioavailable. N-acetyl D-glucosamine binds to glucuronic acid as well as galactose making it a precursor to hyaluronic acid, keratan-sulfate and chondroitin sulfate. This unique derivative aids in proteoglycan, collagen and glycosaminoglycan production. N-acetyl D-glucosamine has also been shown to aid in the healing of soft tissue injury. D-Glucuronic acid is a key substrate comprising one half of the hyaluronan molecule, the other being N-acetyl D-glucosamine.
Supplemental glucosamine has the ability to influence connective tissue such as cartilage, and so may apply to alleviation of various dysfunctions including arthritis. In the joint, for example, chondroitin sulfate acts to stimulate the production of proteoglycans, glycosaminoglycans, and collagen, inhibits degenerative enzymes excreted by the chondrocytes, and synoviocytes, and aids in nutrient transportation within the synovial fluid. Glucosamine, in particular N-acetyl D-glucosamine, increases the synoviocyte and chondrocyte production and subsequent availability of endogenous hyaluronan by the direct in situ inclusion of its prime substrates galactosamine (through chondroitin sulfate assimilation) and N-acetyl D-glucosamine. The exogenous hyaluronan acts to replace depleted endogenous hyaluronan and to lubricate and coat healthy as well as damaged articular tissue during the reparative process.
A GAG composition marketed as a veterinary medical device as POLYGLYCAN (ArthroDynamic Technologies) comprises chondroitin sulfate, N-acetyl D-glucosamine, and hyaluronic acid. Such proteoglycan compositions are described in U.S. Pat. Nos. 6,979,679 and 7,485,629, which are hereby incorporated by reference in their entireties.
Regenerative cells found in multi-cellular organisms are cells capable of promoting tissue repair and regeneration and reducing inflammation. Stem cells are regenerative cells that can differentiate into a diverse range of specialized cell types. The two broad types of mammalian stem cells are: embryonic stem cells that are found in blastocysts, and adult stem cells that are found in adult tissues. The two classical properties of stem cells are self-renewal and potency. Self-renewal refers to the ability to go through numerous cycles of cell division while maintaining the undifferentiated state, and potency refers to the capacity to differentiate into specialized cell types. Potency specifies the differentiation potential of the stem cells to differentiate into different cell types. For instance, totipotent stem cells are cells produced from the fusion of an egg and sperm cell, as well as the first few divisions of the fertilised egg, they can differentiate into embryonic and extra-embryonic cell types. Pluripotent stem cells are the descendants of totipotent cells and can differentiate into cells derived from any of the three germ layers. Multipotent stem cells can produce only cells of a closely related family of cells (e.g. hematopoietic stem cells differentiate into red blood cells, white blood cells, platelets, etc.). Unipotent cells can produce only one cell type, but have the property of self-renewal that distinguishes them from non-stem cells (e.g., muscle stem cells). In addition, a regenerative cell population frequently comprises not only cells but also microsomes released by the regenerative cells that are important in functions such as immunomodulation inside the body.
Progenitor cells refer to immature or partially undifferentiated regenerative cells, typically found in post-natal animals. Like stem cells, progenitor cells have a capacity for self-renewal and differentiation, although these properties may be more limited. Embryonic stem cells are pluripotent and show unlimited capacity for self-renewal. Thus, they are sometimes referred to as true stem cells. In contrast, many cells termed adult stem cells would be better defined as progenitor cells, as their capacities for unlimited self renewal and plasticity have not been comprehensively demonstrated. The majority of progenitor cells are dormant or exhibit little activity in the tissue in which they reside. They exhibit slow growth and their main role is to replace cells lost by normal attrition. However, upon tissue damage or injury, progenitor cells can be activated by growth factors or cytokines, leading to increased cell division important for the repair process. Examples of progenitor cells include satellite cells found in muscle and the transit-amplifying neural progenitors of the rostral migratory stream.
Mesenchymal stem cells (“MSCs”) (i.e., stromal cells) are pluripotent regenerative cells that can differentiate into a variety of cell types. MSCs can be derived from many tissues including bone marrow, adipose, umbilical cord, and dental pulp. MSCs adhere to plastic tissue culture and are commonly selected from more diverse regerative cell populations based on this property of plastic adherence. For example, bone marrow MSCs (BM-MSCs) can be selected from the cell population present in a bone marrow aspirate by culturing the cells that adhere to the plastic tissue culture surface, and adipose MSC (Ad-MSC) can be selected from adipose stromal vascular fraction (SVF) cells in the same manner. Adipose SVF is the non lipid-filled cell population from adipose tissue and contains a high proportion of regenerative cells. Among the cell types that MSCs have been shown to differentiate into in vitro or in vivo are osteoblasts, chondrocytes, myocytes, adipocytes, neuronal cells, and beta-pancreatic islets cells. MSCs provide the supportive structure in which the functional cells of the tissue reside. In addition, MSCs play roles in tissue healing and repair.
Because in adult organisms, MSCs act as a repair system for the body replenishing specialized cells but also maintaining tissue homeostasis and they are capable of differentiating into different types of tissues, they have been utilized in the treatment of, for example, skeletal and connective tissue disorders.
There is increasing evidence that regenerative cells derived from other tissue, such as adipose-derived regenerative cells, are equally or even more capable than bone marrow derived regenerative cells in repairing or alleviating connective tissue dysfunctions. (See, Toghraie et al., “Treatment of Osteoarthritis with Infra-Patellar Fat Pad Derived Mesenchymal Stem Cells in Rabbit,” Knee, 2011, 18(2):71-75; and Frisbie et al., “Evaluation of Adipose Derived Stromal Vascular Fraction or Bone Marrow Derived Mesenchymal Stem Cells for Treatment of Osteoarthritis,” J. Orthop. Res., 2009, 27(12):1675-1680.)
Intra-articular administration of stem and regenerative cells, particularly MSCs from adipose tissue and bone marrow, is increasingly utilized in clinical practice. Current practice is to administer cells suspended in an inactive carrier such as saline or platelet rich plasma, or in a single component non-sulfated GAG, such as hyaluronan. To date, minimal data have been reported as to the effects of GAGs on stem and regenerative cells and on the optimal composition of GAG formulations for combination with stem and regenerative cells for in vitro and in vivo applications.